1. Field of the Invention
Brown rot of potato is caused by Ralstonia solanacearum race 3 biovar (bv) 2. This invention relates to novel PCR primers and the development of real-time PCR assays for the rapid detection of the potato brown rot pathogen R. Solanacearum race 3 bv 2.
2. Description of the Relevant Art
Ralstonia solanacearum, the causal agent of bacterial wilt, infects over 100 plant species (Kelman, A. 1953. North Carolina Agric. Exp. Stn. Tech. Bull. No. 99). The species has been subclassified into races and biovars. R. Solanacearum race 3 bv 2 is a strain that has become adapted to temperate climates (Haywood et al. 1998. In: Bacterial Wilt Disease: Molecular and Ecological Aspects, Prior et al., Eds. Springer Verlag, Berlin, Germany; Stead et al. 1996. In: Conference Proceedings—Brighton Crop Protection Conference—Pests and Diseases 1996, British Crop Protection Council, Farnham, Surrey, United Kingdom, Pages 1145–1152). Other biovars of R. solanacearum can infect potatoes; however, bv 2 is by far the most destructive biovar in temperate areas. The organism has a narrow host range primarily infecting potato (Hayward, A. C. 2000. Ralstonia solanacearum. Encyclopedia of Microbiology, Vol. 4, Second Edition, Academic Press, New York, N.Y.). Brown rot has emerged recently as a serious disease of potato in Western Europe (Stead et al., supra) and R. Solanacearum bv 2 is listed as a zero tolerance quarantine organism in the European Union (EU) (1998. Official J. Eur. Communities L-235: 1–39). In those countries affected by brown rot, the costs of disease surveillance and eradication have become considerable. The pathogen has been reported in potato in Turkey; but it has not yet been observed in potato in the continental U.S. where no regulation in potato currently exists. However, the report of finding bv 2 in geranium in Wisconsin (Williamson et al. 2001. Phytopathology 91: S75) and Pennsylvania (Kim et al. 2002. Phytopathology. 92:S42) could result in movement of the pathogen into potato.
Asymptomatic seed potato tubers, i.e., those having latent infections, are a major factor in the dissemination of R. Solanacearum to new production fields in Europe (Ciampi et al. 1980. Am. Potato J. 57: 377–386). Because pathogen-free seeds are very important for controlling the disease, assays for detecting R. Solanacearum must be very sensitive. In the EU, seed potato tubers must be certified to be free of R. solanacearum using a recommended serological or classical polymerase chain reaction (PCR)-based technique (Official J. Eur. Communities, supra). Sensitivity of the serological (Elphinstone et al. 1996. OEPP/EPPO Bull. 26: 663–678) and PCR (Seal et al. 1993. J. Gen. Microbiol. 139: 1587–1594) techniques are similar ranging from 103–104 cfu/ml in water or potato core tissue extracts spiked with cells of R. solanacearum. The specificity of the classical PCR technique is very high using primers designed from a DNA fragment described by Fegan et al. (1998. In: Bacterial Wilt Disease: Molecular and Ecological Aspects, Prior et al., Eds., Springer-Verlag, Berlin, Germany); however, costs of classical PCR is much greater than real-time PCR due to the need to do a Southern blot analysis to confirm identification of the PCR product (Schaad et al. 1999. Plant Dis. 83: 1095–1100). A real-time PCR assay has been described for the detection of R. solanacearum race 3 bv 2; however, infected tubers were not tested and the sensitivity of the assay was relatively low (Weller et al. 2000. Appl. Environ. Micro. 66 (7): 2853–2858).
R. solanacearum can be considered a major economic threat to United States agriculture; therefore, there exists a need for new technologies to be examined and novel methods to be developed for the detection and identification of the pathogen causing brown rot in potato. If R. solanacearum bv 2 were introduced into potato, all potato shipments would be stopped, resulting in major economic losses. Thus, specific primers and methods capable of identifying latent infections of R. solanacearum race 3 bv 2 in seed potato tubers rapidly and economically are needed.